Carbohydrate-containing external invertase of Saccharomyces cerevesiae will be used as a model system for studying the biosynthesis of a secreted glycoprotein of known composition. Partially glycosylated invertase intermediates in the subcellular rough membrane, internal pool, and plasma membrane fractions will be isolated by immunoprecipitation from extracts of yeast protoplasts labeled with (3H)amino acids and (14C)glucosamine or mannose. Comparative compositional analyses of the recovered material in relation to the secreted holoenzyme should provide information on the sites of carbohydrate attachment and reveal whether oligosaccharide synthesis occurs by transfer to the peptide of large sugar precursors or by many small sequential additions. The kinetics of labeling of specific dolichol intermediates and the presence of sugar transferases in relation to invertase will be examined to clarify the pathway involved in this glycoprotein's synthesis. Additional studies will resolve whether the two glucosamine residues in the di-N-acetylchitobiose moiety in the oligosaccharide core are derived from the same metabolic pool and what effect the glycosylation inhibitors, 2-deoxy-D-glucose and tunicamycin, have on the fate of the invertase peptide. It is planned to apply the results of these studies to the development of an in vitro system capable of the de novo synthesis and glycosylation of yeast invertase.